The ability to deplete specific populations of cells provides a powerful tool for the study of the role of particular cells in, for example, development and other biological processes. For example, the ability to selectively deplete neurons provides neurobiologists tools to study the involvement of different cell types in the functional and structural organization of the nervous system. A number of methods have been devised for this purpose in the context of neuronal cell depletion, including photoablation (He and Masland, 1997 Nature 389:378–382), systemic administration of excitatory neurotransmitters (Johnson and Reese, 2000 Association for Research in Vision and Ophthalmology, Inc. (ARVO), Annual Meeting in Fort Lauderdale, Apr. 30–May 5, 2000, IOVS Abstract Issue 41(4):#4507, Mar. 8, 2000), as well as the administration of immunotoxins directed at specific receptors expressed by targeted neurons (Wiley, 1996 Sem Cancer Biol 7:71–77; Wiley et al. 1997 Neurosci Lett 230:97–1000; Youle et al. 1980 Proc. Natl. Acad Sci USA 77:5483–5486; Flavell, 1998 Curr Top Microbiol. Immun 234:57–61). It is also desirable to delete cells at specific stages of development and to localize effects to a confined region of the nervous system. The effectiveness of a given method is evaluated by its selectivity (e.g., killing of the target cell with little or not killing of non-target cells) and by how completely the method succeeds in eliminating a targeted cell population.
Toxins that can be used to kill cells are generally referred to as cytotoxins. Ribosome inactivating proteins (RIPs), which are a class of proteins ubiquitous in higher plants, are examples of such cytotoxins. RIPs, which are divided into Type I and Type II classes, are cytotoxic due to their activity as potent inhibitors of eukaryotic protein synthesis. Type I RIPS are composed of a single peptide chain having ribosome-inactivating activity, while Type II proteins are composed of an A-chain, essentially equivalent to a Type I protein, disulfide-linked to a B-chain having cell-binding properties. The N-glycosidic bond of a specific adenine base is hydrolytically cleaved by RIPs in a highly conserved loop region of the 28S rRNA of eukaryotic ribosomes, thereby inactivating translation in eukaryotic cells. See, e.g., U.S. Pat. No. 5,744,580. Gelonin, dodecandrin, tricosanthin, tricokirin, bryodin, Mirabilis antiviral protein (MAP), barley ribosome-inactivating protein (BRIP), pokeweed antiviral proteins (PAPS), saporins, luffins, and momordins are examples of Type I RIPs; whereas ricin and abrin are examples of Type II RIPS.
One conventional protocol for constructing an immunotoxin to a targeted cell involves three basic steps: (i) activating the targeting antibody using the heterobifunctional cross-linking agent, N-succinimidyl 3-(2-pyridildithio)propionate (SPDP) to generate sulfhydryl-reactive pyridyldisulfide groups; (ii) modifying the cytotoxin (e.g., a RIP, such as saporin), to contain -SH functional groups; and (iii) mixing the modified antibody and saporin molecules together. The result is a cross-linked antibody-toxin conjugate containing “cleavable” bonds (Carlsson et al., 1978 Biochem. J. 173:723–737). One of the most widely used immunotoxins, 192 IgG-saporin, has been directed at cholinergic neurons in basal forebrain (Robertson et al., 1998 Cerebral. Cortex 8:142–155). The 192 IgG-saporin is targeted to the p75 receptor since it is believed that most cholinergic cells in basal forebrain express this low affinity neurotrophin receptor.
Depletion of cholinergic cells have been of particular interest in the examination of developmental and functional pathways. A common cholinergic cell of interest is the amacrine cell. Cholinergic amacrine cells are arguably one of the best characterized of the more than 40 different amacrine cells thought to be present in the mammalian retina (Vaney, 1990 Prog. in Retinal Res. 9:49–100). These retinal interneurons have been suggested to play several key functions during the development of the retina as well as in the functional organization of the mature retina. A developmental phenomenon ascribed to cholinergic amacrine cells is the generation of retinal waves of activity thought to be crucial for the refinement of patterns of projections in the developing visual system (Burgi and Grzywacz, 1994 J. Neuroscience 14:7246–7439). The use of immunotoxins in research would make it feasible to assess the effects of selectively eliminating these retinal interneurons on the generation of activity patterns in the developing and mature retina.
Unfortunately, use of immunotoxins in various applications (e.g., as research reagents) has been frustrated due to the lack of specificity of the immunotoxin (which can result in killing of or damage to non-target cells) and/or the inefficiency of killing of target cells. For example, the effects of cholinergic cell depletion on the formation of functional, developmental, and structural pathways is a frequent subject of research. However, none of the currently available methods permits selective elimination of the entire population of the targeted cell type. The 192 IgG-saporin immunotoxin in present use is no exception, at least in part because the target site of the immunotoxin, the NGF (p75) receptor, is expressed by several other retinal cell types, including some ganglion cells and Muller glial cells (Hu et al., 1998 Glia 24:187–197). Therefore, other non-target cells nearby are also eliminated. In addition, the effectiveness of 192 IgG-saporin in eliminating cholinergic neurons has been reported to be quite variable at other levels of the nervous system, ranging from just over 40% to more than 90% (Robertson et al., 1998 Cerebral Cortex 8:142–155). Furthermore, the toxins currently employed for eliminating cholinergic cells have serious shortcomings that limit their usefulness. For instance, the AF64A toxin has been found to induce non-specific damage both in the retina as well as at other levels of the central nervous system (McGurk et al., 1987 Neuroscience 22:215–224).
There is a need in the field for immunoselective targeting agents that selectively and efficiently deliver compounds to a target population of neuronal cells, either for elimination of such cells or to promote survival of such cells. The present invention addresses this need.